Porous substrates for DNA arrays

ABSTRACT

A planar, rigid substrate made from a porous, inorganic material coated with cationic polymer molecules for attachment of an array of biomolecules, such as DNA, RNA, oligonucleotides, peptides, and proteins. The substrate has a top surface with about at least 200 to about 200,000 times greater surface area than that of a comparable, non-porous substrate. The cationic polymer molecules are anchored on the top surface and in the pores of the porous material. In high-density applications, an array of polynucleotides of a known, predetermined sequence is attached to this cationic polymer layer, such that each of the polynucleotide is attached to a different localized area on the top surface. The top surface has a surface area for attaching biomolecules of approximately 387,500 cm 2 /cm 2  of area (˜7.5 million cm 2 /1×3 inch piece of substrate). Each pore of the plurality of pores in the top surface of the substrate has a pore radius of between about 40 Å to about 75 Å. Not only does the cationic coating in and over the pores of the substrate greatly increase the overall positive charge on the substrate surface, but also given the size of the pores provides binding sites to which biomolecules can better attach.

[0001] The present Application claims the benefit of priority as acontinuation-in-part to U.S. patent application Ser. No. 09/650,885,filed on Aug. 30, 2000, which claims benefit of priority to U.S. patentapplication Ser. No. 09/562,829, filed on May 1, 2000, now abandoned.The contents of both of the aforementioned patent applications areincorporated herein by reference.

BACKGROUND OF THE INVENTION

[0002] High-density arrays (HDAs) are new tools used by drug researchersand geneticists to provide information on the expression of genes. Ahigh-density array typically comprises between 5,000 and 50,000 probesin the form of single stranded DNA, each of a known and a differentsequence, arranged in a predetermined pattern on a substrate. The arraysare used to test whether single stranded target DNA sequences interactor hybridize with any of the single stranded probes on the array. Thetesting procedure consists of printing and binding single-stranded DNAmolecules onto a substrate. The substrate may be any size, but typicallytakes the form of a standard 1×3 inch microscope slide. The printed DNAsequence is for a known genetic risk factor and may be tagged with afluorescent marker for identification. Unknown, single-stranded DNA,such as obtained from a patient, is tagged with a different fluorescentmarker and washed over the slide for a specified period of time and thenrinsed. If the unknown DNA contains any strands that have complementarynucleic acid sequences to the known strand, hybridization occurs. Anyhybridization on the rinsed slide is detected as fluorescence from themarker on the unknown DNA. Fluorescence above a predetermined, thresholdintensity indicates that the unknown DNA contains that risk factorassociated with the known DNA printed on the slide.

[0003] After exposing the array to target sequences under selected testconditions, scanning devices can examine each location on the array anddetermine the quantity of targets that are bond to complementary probes.The ratio of fluorescent intensity at each spot on the high-densityarray provides the relative differential expression for a particulargene. DNA arrays can be used to study the regulatory activity of genes,wherein certain genes are turned on or “up-regulated” and other genesare turned off or “down-regulated.” So, for example, a researcher cancompare a normal colon cell with a malignant colon cell and therebydetermine which genes are being expressed or not expressed in theaberrant cell. The regulatory cites of genes serves as key targets fordrug therapy.

[0004] Proper performance of a DNA array depends on two basicfactors: 1) retention of the immobilized probe nucleic sequences on thesubstrate, and 2) hybridization of the target sequence to theimmobilized probe sequence, as measured by fluorescence emission fromthe tagged target sequence. The DNA probe material must be retained onthe surface of the substrate through a series of washing, blocking,hybridizing, and rinsing operations that are commonplace in DNAhybridization assays. An excessive loss of probe DNA sequences can leadto a low fluorescent-signal-to noise ratio and uncertain or erroneousresults.

[0005] DNA arrays have for years been printed onto organic, micro-porousmembranes such as nylon or nitrocellulose. The densities at which onecan print DNA solutions onto these types of organic micro-porousmembranes is limited because of the tendency for the DNA solution towick laterally through the membrane, thus causing cross-talk andcontamination between adjacent locations. Others have employed a flat,non-porous substrate surface made from glass. (See for example, U.S.Pat. No. 5,744,305, incorporated herein by reference.) These substrates,however, have also been found wanting, since they do not retain theprobe molecules as well as porous substrates.

[0006] The present invention proposes to use a substantially flat,porous, inorganic substrate surface that is specially treated withcationic polymer coating to enhance retention of nucleic moieties forhigh-density arrays. The porous surface provides increased surface areafor immobilizing DNA probe molecules, which increases the density of DNAbinding sites per unit cross-sectional area of the substrate. Theincreased number of possible binding sites per unit area results ingreater retention of immobilized DNA probes and the emission of anincreased signal when hybridized with target molecules.

SUMMARY OF THE INVENTION

[0007] The present invention relates to a biological analysis device,which comprises a planar, rigid substrate made from a porous, inorganicmaterial with a top surface having about at least 200 to about 200,000times greater surface area than that of a comparable, non-poroussubstrate. The substrate can be employed to fabricate high-densityarrays. A layer of cationic polymer molecules is anchored on the topsurface and in the pores of the porous material. The cationic polymersallow for the attaching of an array of biomolecules such as DNA, RNA,oligonucleotides, peptides, and proteins. In high-density applications,an array of polynucleotides of a known, predetermined sequence isattached to this cationic polymer layer, such that each of thepolynucleotide is attached to a different localized area on the topsurface.

[0008] The top surface has a surface area for attaching biomolecules ofapproximately 387,500 cm²/cm² of area (˜7.5 million cm²/1×3 inch pieceof substrate). Each pore of the plurality of pores in the top surface ofthe substrate has a pore radius of between about 40Å to about 70 Å. Thetop surface is composed of a borosilicate glass. A layer of cationicpolymer is applied and attached either by electrostatical means or bymeans of dip-coating to the top surface. The cationic polymer is, forinstance, either polylysine, polyethylene-imine, polybrene, orγ-amino-propyltriethoxysilane. Not only does the cationic coating in andover the pores of the substrate greatly increase the overall positivecharge on the substrate surface, but also given the size of the poresprovides binding sites to which biomolecules can better attach.

[0009] The invention also relates to a method of preparing a porousglass substrate for attaching biomolecules. The method comprises thesteps of providing a porous glass having pores each between about 40Å toabout 75Å in size; applying electrostatically a layer of cationicpolymer to a top surface of said glass at an acidic pH value—typically apH of about 0 or 1-6; then washing and drying the glass; and heating theglass to about 140-175° C. to cross-link free silanol groups. Thecationic polymer molecules coat not only the top surface of the poroussubstrate, but also the surfaces of the plurality of interconnectedchannels and voids that extend within the substrate.

BRIEF DESCRIPTION OF THE DRAWINGS

[0010]FIG. 1 is a schematic cross-sectional representation of a poroussubstrate of the present invention with double stranded DNA moleculesattached to the substrate by means of electrostatic interaction with acationic polymer.

[0011]FIG. 2 is a fluorescence scan image of spotted single strandedCy3-labeled DNA on a porous glass substrate according to the presentinvention.

[0012]FIG. 3 is a fluorescence scan image of spotted single strandedCy3-labeled single-stranded DNA that has hybridized to an immobilized,complimentary single stranded DNA sequence.

DETAILED DESCRIPTION

[0013] Open celled porous glasses having the desirable properties offused silica have been applied for various uses. Such glasses areproduced using a unique process that circumvents the needs for hightemperatures in melting and forming, which affords cost andmanufacturing savings. According to the process, a relatively softalkali borosilicate glass preform is melted in a convention manner andis then pressed, drawn or blown into the desired shape by standardprocesses used in glass production. The resulting workpiece, whichoccasionally is given additional finishing operations, is subjected to aheat treatment above the annealing point but below the temperature thatwould produce deformation of the glass. Two continuous closelyintermingled glassy phases are produced during the heat treatment. Onephase, which is rich in alkali and boric oxide, is readily soluble withacid. The other phase, which is rich in silica, is insoluble. After theworkpiece is immersed in a hot dilute acid solution, the soluble phaseis dissolved, leaving behind a fine, porous, high-silica lattice orspider-web-like shell (96% silica). The resulting porous silica articleis commonly known as thirsty glass (commercially known as Vycor®, Code7930, by Corning Inc.).

[0014] Porous glasses, like Vycor®, are mechanically strong, hard,non-flaking, and chemically inert. The open network permits selectivepermeability. Pore size in the glass varies, generally ranging between40-200 Å, but are preferable used from 40 Å to 60 Å or 70 Å. Pore sizedistribution in a piece of glass is typically very narrow (±3 Å fromaverage pore radius). The pore size may be adjusted as desired, forexample, by dissolving the glass with a weakly reactivefluorine-containing compound. See generally, T. H. Elmer, “Porous andReconstructed Glasses,” ENGINEERED MATERIALS HANDBOOK, Vol. 4, Ceramicsand Glasses, pp. 427-32, ASM International (1992), incorporated in itsentirety herein by reference.

[0015] Due to its porosity, a material like Vycor® has an internalsurface area of approximately 250 square meters per gram. A Vycor® glassslide weighing approximately 3 grams has ˜7.5 million cm² of 1×3 inchsurface area (alternatively, 2.5 million cm²/cm²). Comparatively, anon-porous glass slide of the same dimensions has only about 40 cm² ofsurface area—a difference of a factor of 200,000 for biomoleculeattachment. Alternatively, the porous substrate can be characterized ashaving a plurality of interconnected voids of a predetermined mean sizeof about 40 Å or 50 Å dispersed therethrough, and having void channelsthat extend through to a top surface of the porous substrate.

[0016] According to the present invention, a porous glass slide is usedas a substrate for immobilizing biomolecules, in particular DNA (e.g.,cDNA or oligonucleotides). The porous inorganic substrate of the presentinvention can be treated with cationic polymers (i.e., polymers having amultiplicity of ionic or ionizable functional groups having a positivecharge) to advantageous effect. A non-exhaustive list of examples ofcationic polymers that may be suitable for use as coatings on the porousglass substrate include: polylysine and corresponding copolymers withneutral amino acids, polyethylene-imine, polybrene, aminosilanes such asγ-amino-propyltriethoxysilane (GAPS), cationic dendrimers or starpolymers, and polyvinylamine.

[0017] Coating a porous inorganic substrate with cationic polymers hasseveral advantages over cationic coatings on flat nonporous substrates.First porous glass substrates have a greater surface area and thereforehave more surface-exposed silanol groups. Consequently, the negativelycharged silanol groups can bind to greater amounts of the positivelycharged cationic species (e.g., amino silanes, polylysine, cationicdendrimers) by electrostatic interactions. This greater density ofpositive charge lends to greater retention of the negatively charged DNAmolecules.

[0018] Second, the electrostatic interaction between DNA molecules andcationic polymer coating is stronger for a porous glass substrate thanthe same interaction on a nonporous glass substrate because of thegreater local availability of exposed silanol groups for electrostaticbinding per cationic polymer molecule. Further, the narrow pore sizes(˜40-75 Å) create an environment of tightly bound water molecules whichproduces lower dielectric constants. This microenvironment is likely togreatly enhance the strength of the electrostatic interactions betweensilanol groups and cationic polymer molecules.

[0019] Third, displacement of the cationic polymer from the upper-mostsurface of the porous glass substrate does not necessarily result incomplete displacement of the polymer from the substrate. As illustratedin FIG. 1, the cationic polymer molecules 12 remain attached to thesubstrate 10 by its tail within pores 14 even if most of the polymercoating is removed from the surface 16. It is also thought that thistype of dynamic equilibrium can enhance DNA hybridization by serving toextend the probe DNA strand 18, which is bound to the polycation 12, offof the substrate surface 16 into solution, thus eliminating stericinterference to hybridization by the substrate.

[0020] To facilitate the entry of cationic polymer further into thepores of the particular substrate described above, the polymer is firstdissolved in a buffer at a pH above the pK_(a) of the amino group. Thisprevents electrostatic bonding of the polymer to the flat upper surfaceof the substrate. Only after the pH is equilibrated with the poroussubstrate is the pH is lowered to make the polymer bindelectrostatically. A substrate prepared according to this fashion shouldhave a higher signal to noise (S/N) ratio than conventional primarycoatings consisting of aminosilanes (e.g., CVD GAPS) on poroussubstrates. The pores of the substrate would be sterically blocked bythe cationic polymer, leading to reduced amounts of non-specific bindingof DNA to the substrate.

[0021] Moreover, the cationic polymer-coated porous glass substrate ofthe present invention is compatible with existing probe-retention andtarget DNA-hybridization protocols. For instance, addition of probe DNAcan be followed by blocking of the substrate with a deactivator (e.g.,succinic anhydride or anionic polymers: polyglutamic acid, polyacrylicacid, anionic dendrimers, heparin, etc.), which would confer a netnegative charge to the substrate surface. This modification of thesubstrate would reduce non-specific binding of target DNA strands andreduce background signal either by electrostatic or steric repulsion.

EXAMPLE

[0022] A number of 1×3 inch×1 mm porous glass slides (Vycor®, CorningCode 7930) were coated with gama-amino-propyltriethoxylsilane (GAPS)using a 1.0% aqueous solution of GAPS at a pH of 4.0, adjusted usingacetic acid. Generally, the treatment can be applied at any acidic pHvalue. Applicable pH values will range typically between about 5 or 6 toabout 2 or 1, more preferably about 3 or 4. The coatings were done bycompletely immersing each slide in the silane solution (dip-coating) forabout 30 minutes. The coated slides were then washed thoroughly withdistilled water and dried. The slides were then heated to 160° C. inorder to cross-link any free silanol groups. Temperatures can range fromabout 140-175° C., but more typically between 150-170° C.

[0023] Next, a water and glycerin solution containing Cy3-labeled singlestranded 80-mer oligonucleotides in a concentration of 1 picomole oligoper microliter was printed using a micropipette onto the treated slidesurface. The printed slide was heated in humidity for one hour at 55° C.The slides were then washed twice with 5×SSC and 0.1% SDS at 55° C. toremove any unattached or unbonded DNA. The slides were then treated witha solution of succinic anhydride dissolved in DMF for blocking.

[0024] The single stranded DNA immobilized on the blocked slides werenext hybridized with Cy5-labeled complimentary DNA sequences at 55° C.using a probe-clip press-seal incubation chamber in a hybridizationbuffer (Boehringer Mannheim, Cat. # 1717473). The slides were thenscanned using a fluorescence detection scanner (Scan Array 3000, GeneralScanning Inc.). The slides were scanned twice. First, the slides werescanned for Cy-3 prior to hybridization with the complimentaryoligonucleotides, and again for Cy-5 after hybridization.

[0025] Results

[0026]FIG. 2 shows a Cy-3 scan of a sample slide 2 prior tohybridization, and after the two washings with 5×SSC and 0.1% SDS at 55°C. As shown, the signal strength from the four spotted regions 5,indicates significant oligonucleotide immobilization. FIG. 3 shows a Cy5scan of the same sample slide 2 after hybridization with complimentarylabeled oligonucleotides. As shown, the signal strength from the spottedregions 5 indicates detectable hybridization. This event suggestscontinued retention of the probe to the porous glass surface even afterblocking and washing steps.

[0027] The present invention has been described by way of example, andthose skilled in the art will understood that the invention is notnecessarily limited to the embodiments specifically disclosed, and thatvarious modifications and variations can be made without departing fromthe spirit and scope of the invention. Therefore, unless changesotherwise depart from the scope of the invention as defined by thefollowing claims, they should be construed as included herein.

We claim:
 1. A biological analysis device comprising: a planar, rigidsubstrate made from a porous, inorganic material, said substrate havinga top surface with about at least 200 to about 200,000 times greatersurface area than that of a comparable, non-porous substrate, and havinganchored on said top surface and in the pores of said porous materialcationic polymer molecules for attaching an array of biomolecules byelectrostatic means.
 2. The biological analysis device according toclaim 1, wherein said top surface has a surface area for attachingbiomolecules of approximately 387,500 cm²/cm² of area.
 3. The biologicalanalysis device according to claim 1, wherein said top surface hasapproximately 7.5 million cm² of surface area for attaching biomoleculesin a 1×3 inch piece of substrate.
 4. The biological analysis deviceaccording to claim 1, wherein said top surface has a surface area ofapproximately 2.5 million cm²/cm² of area.
 5. The biological analysisdevice according to claim 1, wherein said layer of cationic polymer isapplied and attached electrostatically to said top surface.
 6. Thebiological analysis device according to claim 1, wherein said layer ofcationic polymer is applied by means of dip-coating.
 7. The biologicalanalysis device according to claim 1, wherein said cationic polymerincludes at least one of the following: polylysine, copolymers withneutral amino acids, polyethylene-imine, polybrene, aminosilanes,cationic dendrimers, star polymers, and polyvinylamine.
 8. Thebiological analysis device according to claim 1, wherein said cationicpolymer is either polylysine or polyethylene-imine.
 9. The biologicalanalysis device according to claim 1, wherein said cationic polymer isγ-amino-propyltriethoxysilane.
 10. The biological analysis deviceaccording to claim 1, wherein said biomolecules comprise DNA, RNA,oligonucleotides, peptides, and proteins.
 11. The biological analysisdevice according to claim 1, wherein said porous substrate is furthercharacterized as having a plurality of interconnected voids of apredetermined mean size of between about 40-75 Å dispersed therethrough,and having void channels that extend through to a top surface of theporous substrate.
 12. The biological analysis device according to claim5, wherein each of said voids is about 40 Å to about 60 Å in size. 13.The biological analysis device according to claim 1, wherein said topsurface is composed of a borosilicate glass.
 14. A device for performingmultiple assays, the device comprising: a) a porous inorganic substratehaving a planar top surface, said substrate having a top surface with asurface area of from about 200 to about 200,000 fold greater than acomparably sized, non-porous substrate; b) a cationic polymer layercomprising at least one of the following: polylysine, copolymers withneutral amino acids, polyethylene-imine, polybrene,γ-aminopropyltriethoxysilane cationic dendrimers, star polymers, andpolyvinylamine bonded to said top surface; c) an array ofpolynucleotides of a known, predetermined sequence attached to saidcationic polymer layer, whereby each of said polynucleotide is attachedto a different localized area on said surface.
 15. The device accordingto claim 13, wherein said top surface has approximately 7.5 million cm²of surface area in a 1×3 inch piece of substrate.
 16. The deviceaccording to claim 13, wherein said porous substrate is furthercharacterized as having a plurality of interconnected voids of apredetermined mean size of between about 40-70 Å dispersed therethrough,and having void channels that extend through to a top surface of theporous substrate.
 17. A method for preparing a porous glass substratefor attachment of biomolecules, the method comprising: providing aporous glass having pores each between about 40 Å to about 75 Å in size;applying electrostatically a layer of cationic polymer to a top surfaceof said glass at an acidic pH; washing and drying said glass; heatingsaid glass to about 140-175° C. to cross-link free silanol groups. 18.The method according to claim 17, wherein said pH value is between about1-6
 19. The method according to claim 17, wherein said pH value isbetween about 3-5.
 20. The method according to claim 19, wherein said pHvalue is 4.0.
 21. The method according to claim 17, wherein saidtemperature to which said glass is heated is about 150-170° C.
 22. Themethod according to claim 17, wherein said layer of cationic polymer isapplied by means of dip-coating.
 23. The method according to claim 17,wherein said cationic polymer includes at least one of the following:polylysine, copolymers with neutral amino acids, polyethylene-imine,polybrene, aminosilanes, cationic dendrimers, star polymers, andpolyvinylamine.
 24. The method according to claim 17, wherein saidcationic polymer is either polylysine or polyethylene-imine.
 25. Themethod according to claim 17, wherein said cationic polymer isγ-aminopropyltriethoxysilane.
 26. The method according to claim 17,wherein said biomolecules comprise DNA, RNA, oligonucleotides, peptides,and proteins.
 27. A substrate for a biological analysis device madeusing the method according to claim 17.